Phylogenetic relationships of sleeper gobies (Eleotridae: Gobiiformes: Gobioidei), with comments on the position of the miniature genus Microphilypnus

Microphilypnus and Leptophilypnion are miniaturized genera within the family Eleotridae. The evolutionary relationships among these taxa are still poorly understood, and molecular analyses are restricted to mitochondrial genes, which have not been conclusive. We compiled both mitochondrial and nuclear genes to study the phylogenetic position of Microphilypnus and the evolutionary history and relationships of eleotrids. We propose that Microphilypnus and Leptophilypnus (a non-miniature genus) are not sister groups as suggested by previous studies, but rather separate lineages that arose in the early Eocene, with Leptophilypnus recovered as a sister group to the other analyzed eleotrids. In fact, Microphilypnus is currently associated with the Neotropical clade Guavina/Dormitator/Gobiomorus. We also identified a well-supported clade that indicated Gobiomorus and Hemieleotris as paraphyletic groups, besides a close relationship among Calumia godeffroyi, Bunaka gyrinoides, Eleotris and Erotelis species. This is the first comprehensive report about the evolutionary relationships in members of the family Eleotridae, including multiloci and multispecies approaches. Therefore, we provided new insights about the phylogenetic position of some taxa absent in previous studies, such as the miniature genus Microphilypnus and a recently described species of Eleotris from South America.


Miniaturization in eleotrids.
Miniaturization is an evolutionary process that leads to reduced body size of lineages over time, being observed in several groups of fishes, amphibians, reptiles, and primates [26][27][28] . In general, miniaturization is accompanied by structural simplifications, novel structures, and increased variation 29 . In some cases, the truncated development eventually determines the appearance of distinct evolutionary novelties, including "bizarre" forms 30,31 . The miniature taxa (traditionally defined as those species with a total length below 25 mm) are particularly diversified in ichthyofauna 32 . For example, only in the Neotropical region, nearly 210 miniature fish taxa have been reported encompassing the main orders: Characiformes, Siluriformes, Cyprinodontiformes, Perciformes, and Gobiiformes 33 . In the order Gobiiformes, two miniature genera of the family Eleotridae are recognized, namely: Microphilypnus [34][35][36][37] , and Leptophilypnion 38 .
Although the description of miniature forms and their adaptive relationships have been addressed since classical works by Haeckel 39 , current phylogenetic approaches might elucidate the tempo and mode of evolution in body size of species and/or lineages [40][41][42] . However, the few studies available based on molecular data involving the miniature genera Microphilypnus have generated contrasting evolutionary scenarios. The authors 7,11 hypothesized a phylogenetic relationship between miniaturized species of the genus Microphilypnus and with non-miniaturized genus Leptophilypnus from coastal rivers in Central America. On the other hand, Thacker 43 demonstrated that Microphilypnus is more closely related to another non-miniature genus (Philypnodon), endemic to freshwater ecosystems in Australia. Therefore, further investigations are required to provide a reliable phylogenetic reconstruction about the evolutionary transition from non-miniature to miniature groups, and thus, determine whether miniaturized genera represent a miniaturized clade or the result of independent evolutionary events of miniaturization.
Therefore, to refine the evolutionary relationships within Eleotridae, we generated a more comprehensive phylogeny of this family based on mitochondrial and nuclear genes, including taxa excluded from previous phylogenetic reports and comprising different biogeographic regions. Although molecular data from representatives of the miniature genus Leptophilypnion are still absent, our multi-locus phylogenetic analyses allow exploring the phylogenetic position of the miniaturized group Microphilypnus, which has often been neglected in former studies.

Results
The final dataset in molecular phylogenetic analyses consisted of 52 taxa, being four of them related to outgroups. The final concatenated alignment of mitochondrial and nuclear DNA sequences had a length of 3494 bp (16S, COI, ND2, Rhod, EGR1) and the phylogenetic reconstruction was based on the Bayesian coalescence approach (species tree on *BEAST) (Fig. 2). The two exons used had a total of 247 variable sites in 1240 bp (EGR1 = 118/811, and Rhod = 129/429). The dataset partitioning scheme and the nucleotide substitution models for multilocus phylogenetic analyses are shown in Supplementary Table S2. The phylogenetic reconstruction confirmed the monophyly of the family Eleotridae and revealed six well-supported main clades (> 0.99 PP): (1) Eleotris/Erotelis/Bunaka/Calumia; (2) (6) genus Leptophilypnus (Fig. 2).
The first clade (in blue) includes Eleotris from Neotropical and Indo-Pacific regions. The relationships between Bunaka gyrinoides and Calumia godeffroyi and the species of the genus Eleotris and Erotelis were strongly supported (PP > 0.99). We found that Eleotris species from the western Atlantic (E. amblyopsis, E. pisonis, E. perniger, and the recently discovered lineages but not formerly described (Eleotris sp. 1 and Eleotris sp. 2) form a monophyletic group. From the biogeographic point of view, our phylogenetic analyses showed that the neotropical species are not monophyletic (blue, green and red clades). Instead, the clade from Australia and New Zealand (yellow clade) is a sister group of the Eleotris lineage (PP = 0.99). In this clade, Gobiomorphus (represented by species from Eastern Australia and New Zealand) was recovered as a sister group of Philypnodon (Eastern Australia). Also, the close relationship between Guavina and Dormitator (Neotropical region) was well supported (PP = 1.0), while Dormitator latifrons (from Eastern Pacific) has been recovered as a sister lineage in relation to D. maculatus (Western Atlantic), D. cubanus (Cuba) and D. lebretonis (Western Africa).
The STACEY and SpeciesDA analyses considering all species involved in this study using all the molecular data (both mtDNA and nDNA) recovered strong support (see Supplementary Fig. S1 online) for a species delimitation hypothesis in which all putative taxa within the species group were distinct. Besides, the multi-locus approach (STACEY), also recognized the same relationships demonstrated between the more internal clades, which reinforces the robustness of our results in the face of phylogenetic uncertainties evidenced in previous works.
The miniature fish of the genus Microphilypnus were placed in a phylogenetic framework with eleotrid species from different biogeographic regions, suggesting a close evolutionary relationship with high support values (PP > 0. 98

Discussion
The present study consists of a robust phylogenetic reconstruction of the family Eleotridae based on multiple loci (mtDNA and nuDNA). Based on these results, we inferred phylogenetic hypotheses to shed light on the evolutionary history of freshwater and estuarine eleotrids, encompassing the evolutionary relationships among 48 species. Although multilocus analyses have been performed recently, some important questions remain unresolved. For example, McCraney et al. 14 showed interesting results on the phylogenetic relationships of the large Gobiaria group but indicated some instability within the Eleotridae. The authors do not discuss the causes of www.nature.com/scientificreports/ this instability in detail, but probably they can be explained by complex evolutionary scenarios. Furthermore, in the broad phylogeny of McCraney et al. 14 ancestral relationships within the Eleotridae family are uncertain. In this study, it is not possible to determine ancestral relationships between Eleotris/Gobiomorphus/Dormitador/G uavina (although this uncertainty extends to other clades within the family). Therefore, to resolve the remaining controversies from previous studies, in addition to using species not included in previous phylogenies, we focused specifically on the family Eleotridae, which simplifies the evolutionary reconstruction scenario. In this way, we believe that the results obtained here can help to clarify some of these points. Miniaturization is a recurrent theme in evolutionary studies since this phenomenon involves processes related to the reduction of body size usually associated with remarkable changes in morphology, physiology, ecology, life history, behavior, and reproductive maturity of organisms 29 . From a genetic point of view, phylogenetic approaches can greatly contribute to unraveling the evolution of miniature species. For example, the phylogenetic position of the miniature genus Paedocypris, considered one of the smallest groups of vertebrates (standard length of 10-12 mm), was determined based on inferences from mitochondrial DNA (cytochrome b) 40 . These authors located Paedocypris as a sister group to the miniature species of the genus Sundadanio, both of which were found to be sister lineages and the other taxon within the family Cyprinidae. Later, Britz et al. 41 provided a more consistent phylogenetic signal of this group based on six nuclear genes, where Paedocypris appears as a sister group to all cyprinids. Both reports indicated that the miniaturization processes have taken place independently.
In the case of Eleotridae, previous phylogenetic reconstructions based on mitochondrial genes corroborated Microphilypnus and the non-miniature genus Leptophilypnus as sister groups 7,11,12 , (Fig. 1). Here, we found strong support in the species tree that included the miniature species of Microphilypnus within the Neotropical clade Dormitator/Guavina/Gobiomorus/Hemieleotris ( Fig. 2; Supplementary Fig. S1 online). However, as we did not include miniaturized Leptophilypnion representatives in our phylogeny, we cannot claim that Leptophilypnion is the sister group of Microphilypnus, and that miniaturization arose once in a clade, or twice independently throughout the evolution of the Eleotridae.
Apart from the fact that the phylogenetic position of the genera Microphylipnus and Leptophylipnus is still unclear (especially whether they are sister groups or not), no synapomorphy has yet been described for either taxon. Indeed, Microphylipnus exhibits a suite of morphological characters not found in Leptophilypnus and most other eleotrids, such as a reduction in pectoral fin rays (11-15 vs. 15 or more), a barely ossified lateral ethmoid (vs. ossified and conical in frontal view), and a no ossified adult scapula (vs. ossified). Actually, Lepthophylipnus shares some reducing features with Microphilypnus, such as the slender infraorbital region and the absence of a row of infraorbital papillae. However, it is likely that these features evolved repeatedly in both lineages as the result of independent miniaturization events.
It is noteworthy that miniaturization events are often reported in Gobiiformes, suggesting a trend in this group towards the reduction of body size and loss of some morphological traits associated with miniature forms. Besides, the parallel adaptive evolution to similar microhabitats eventually leads to homoplasy, thus hindering the establishment of reliable phylogenetic relationships based only on morphology. On the other hand, the emergence of miniature and phylogenetically divergent groups supports our hypothesis that the miniaturization processes in Eleotridae represent independent evolutionary pathways.
Unfortunately, the phylogenetic position of Leptophilypnion, a recently described genus of Neotropical miniature eleotrids 38 , remains obscure. According to morphological traits, Leptophilypnion would be more related to Microphilypnus than to Leptophilypnus, by sharing some features such as the reduction in the number of scales and pectoral fins. Nonetheless, Leptophilypnion is distinguished by the presence of elongated pelvic fin rays, five branchiostegal rays (vs. six in other eleotrids), and additional unusual characters in skeleton 38 . In this case, inferring the phylogenetic position of Leptophilypnion would be of great importance to elucidate the evolutionary relationships among these species within Eleotridae, specially to clarify a question that remains uncertain, namely, whether miniaturization events within eleotrids arose independently or consisted of ancestral traits. However, after numerous collections at the sites where the holotypes were found (Negro and Tapajós Rivers-Brazil, according to Roberts (2013) 38 , we were unable to find the specimens of the two valid Leptophilypnion species. Alternatively, we tried through partnerships with ichthyological collections and other research groups to obtain these species, but unfortunately, we were not successful. Therefore, more information is needed to clarify the evolutionary relationships between species in these groups.
Eleotris corresponds to the only genus of Eleotridae that is widely distributed in different biogeographic areas, from the Neotropics, Africa, Indo-Pacific to Oceania. Differently to the previous reports 7-11 , which found a close relationship between Eleotris amblyopsis and Eleotris fusca, the new taxa included in our phylogenetic analyses indicated that Eleotris amblyopsis is the sister species of Eleotris sp.2, a newly discovered lineage in Nothern coast of Brazil 6 . We also recovered B. gyrinoides and C. godeffroyi within the clade Eleotris/Erotelis. Both species are distributed in the Indo-Pacific region and have a disjunct range when compared to the Neotropical genera Eleotris and Erotelis. Based on the presence of 10 + 15 vertebrae and pterygiophores of first dorsal fin beginning on the third interneural space, in a series of 1, 2, 2, and one element respectively (combination 3(1221)), the genera Eleotris, Erotelis and Calumia had been referred to the group "Eleotris" 44 , which also includes freshwater and estuarine species of Belobranchus from Indo-Pacific. However, to fathom the fascinating evolutionary history of these genera of eleotrids, robust approaches are needed to determine evolutionary diversification and its relationships to past environmental conditions. For example, the role of dispersal and/or vicariant events in the distribution and phylogeographic structure of these species should be carefully investigated.
Our data revealed a close phylogenetic relationship between the genera Guavina and Dormitator, which has also been reported in previous studies 7, 11,12 . Morphological evidence also supports these results since both genera share one unambiguous synapomorphy first two hemal spines curved, arched (see Birdsong 44) . On the other hand, D. latifrons and D. maculatus were not recovered as sister species, thus differing from previous reports 8, 11 www.nature.com/scientificreports/ species from the Atlantic Ocean, following the same trend observed in the diversification of Eleotris, in which the Atlantic species (D. maculatus, D. cubanus and D. lebretonis) form a monophyletic group. Therefore, our results corroborate the previous inference by Galván-Quesada 46 .
According to the present molecular analyses, Gobiomorus is paraphyletic in relation to H. latifasciata, as also indicated by Thacker 11 . Gobiomorus dormitor (Western Atlantic) and G. polylepis (Eastern Pacific) are also sister species, representing a didactic example of geminate species that diverged after the formation of the Isthmus of Panama. The origin of Eleotridae dates to Eocene (55.6 My), but their ancestral area remains unknown because sister groups to this family were not included in this study. Our phylogenetic analysis successfully recovered the species from Eastern Pacific and Western Atlantic (Erotelis armiger/E. smaragdus; Guavina guavina/G. micropus; Gobiomorus polylepis/G. dormitor; Leptophilypnus fluviatilis / L. panamensis) as sister groups, similarly to the results obtained by Thacke 45 . The time-calibrated phylogeny showed that the lineages diverged before the formation of the Isthmus of Panama 3.1 Mya 47 . However, the most recent speciation events (1.4 Mya) occurred between D. cubanus, endemic to Cuba, and D. lebretonis from "Western-Central Atlantic.
Regarding the miniaturized clade Microphilypnus, the estimate of divergence time of the clade Dormitator/Guavina was approximately 16.6 Mya. Lovejoy et al. 48  Similarly, the paraphyletic status of Gobiomorphus in relation to Hemieleotris herein observed agrees with other reports. However, we suggest caution before the synonymization of these lineages since unambiguous synapomorphies for the clade Hemieleotris and G. polylepis/G. dormitor have not been described. In this context, subsequent radiations throughout the South American basins determined a profusion of morphologically and ecologically distinct species not seen in marine habitats 56 . Exploring this biogeographic background is highly recommended to understand the miniaturization process in Eleotridae. In fact, smaller body sizes in freshwater taxa when compared to marine forms have been widely reported, with explanations ranging from the advantages of reduced size in offering greater maneuverability in structured environments 57 to the reduction of energetic demands in size-constrained or complex microhabitats 31 .
In summary, these data provide the most complete hypothesis for Eleotridae phylogeny to date, because it includes representatives from several biogeographical regions. Our results were based on evolutionary information from mitochondrial and nuclear genes, and then, revealed a novel phylogenetic relationship from previous studies based only on mtDNA. The miniaturization does not seem to be a frequent event in Eleotridae, because the miniature taxa evolved in at least two genera (Microphilypnus and Leptophilypnion). As a result, we propose that miniaturization is an evolutionary process in the genus Microphilypnus with a strongly supported sister group relationship between Microphilypnus and the neotropical genus Guavina, Dormitator and Gobiomorus. As the position of Leptophilypnion was not established in the phylogeny, we cannot affirm the close relationship between the miniature taxa. Thus, more extensive taxonomic and geographical sampling and analysis based on multi loci may reveal whether this event is exclusively part of a clade. The non-miniature genus Leptophilypnus was often considered to be a sister group of the Microphilypnus, however, our results are consistent with the hypothesis that both lineages evolved independently.

Material and methods
Taxon sampling. A total of 48 samples were included in the phylogenetic analyses, being 22 of them collected in the wild and 26 obtained from NCBI GenBank (Table S1). The dataset consisted of 22 species of Eleotridae found exclusively in the Neotropical region, including members of all currently recognized genera, except Leptophilypnion 38 , which has a relatively recent description with no genetic data available in public databases.
To explore the phylogenetic relationships hypothesized in the previous studies 7,11,46 , we also included DNA sequences of species from other biogeographical regions, such as Indo-Pacific, Australia, New Zealand, New Guinea, Madagascar, and Africa. Four species of Gobiiformes (Perccottus glenii, Odontobutis potamophila, Odontobutis obscura and Rhyacichthys aspro) were used as outgroup. All newly acquired sequences were deposited in GenBank (accession numbers in Supplementary Material 1). For the mtDNA markers, the PCRs conditions were as follows: initial denaturation at 94 °C for 4 min, followed by 35 cycles of 40 s at 94 °C, 40 s of annealing, 72 °C for 3 min, and a final extension of 5 min at 72 °C. The amplification conditions for nuDNA included an initial denaturation at 94 °C for 5 min, followed by 40 cycles of 94 °C for 40 s for denaturation, 30 s at 50 °C for annealing (16S, ND2, and COI), and 40 s at 52 °C for annealing (RHOD and EGR1), and 72 °C for 90 s for extension, plus a final extension of 7 min at 72 °C. The efficiency of amplification via PCR was checked in a 2% agarose gel. Amplified products were purified with PEG (polyethylene glycol) and sequencing reactions were performed with the BigDye reagent kit. The purified samples were then sequenced by the Sanger method 61 using an ABC 3500xL automatic sequencer (Applied Biosystems).

Ethical statement.
Phylogenetic analyses. The sequences were aligned automatically using MUSCLE 62 , as implemented in GENEIOUS 9.0.5 (https:// www. genei ous. com). The phylogenetic analyses were performed based on concatenated mitochondrial and nuclear partitions but applying separate priors. The aligned sequences of multiple loci were concatenated using SequenceMatrix 1.7.8 63 . The best-fit evolutionary model was selected in Partition-Finder 2 64 for each gene and for each codon position in the case of protein-coding genes. The best-fit partitioning schemes and models are shown in Supplementary material 2.

Estimates of divergence times and species tree. The analysis of TMRCA (Time of the Most Recent
Common Ancestral) as well as a species tree (*BEAST) 65 were implemented in *BEAST 2.5.2 66 . We used the five genes according to the optimal partitioning strategy as indicated by PartitionFinder 2 (Table S2). The simulations were carried out assuming an uncorrelated lognormal relaxed molecular clock, and the Yule speciation process as a prior 67 . The BEAST analysis comprised two independent runs, using 10 million generations, sampled every 5000 generations. The first 10% of all samples were removed as burn-in, and Tracer 1.7.1 68 was used to check the effective sample sizes (ESS) assuming optimal parameters (> 200). The maximum credibility tree was generated in TreeAnnotator v1.6.1 69 . The resulting phylogenetic trees were visualized in Figtree 1.4.3 70 . The TMRCA was estimated based on the recovered ages in the study developed by Betancur-R 71 . These authors calibrated points from the fossil record using a subset of 202 taxa, 18 genes, and 59 calibration points. Based on this study, we used the origin of the family Eleotridae (mean age of 55.47 Ma) as a calibration point.
To validate the multilocus phylogenetic taxonomy, we performed an analysis in STACEY 1.2.5 72 implemented in Beast 2.6.2. We conducted STACEY analysis using the previously described StarBEAST2 dataset, with all taxa and partitions conserved in both analyses (Supplementary Table S2). Final phylogenetic relationships were estimated in four independent runs for the whole data set. Each run consisted of 50 million iterations and parameter estimates sampling every 10,000 generations, discarding the first 10% as burn-in. STACEY log files were examined in Tracer v.1.7.1 67 to assess whether the runs have reached the stationary phase and converged on model parameters (ESS > 400). Support of topologies was evaluated in STACEY by constructing a tree of maximum reliability in TreeAnnotator after the rejection of half of all estimated trees. Species delineation (based on the trees evaluated in STACEY) was carried out using a Java-application speciesDA (http:// www. indri id. com/ softw are. html), using simcutoff 1 and collapse height 0.0003.

Data availability
The datasets generated and analyzed in the current study are available in GenBank (GenBank accession numbers are shown in supplementary material). www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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